Synaptic transmission is a highly regulated process that mediates information processing in the brain. Disturbances in synaptic transmission are associated with numerous neuropsychiatric disorders from Parkinson's disease and epilepsy to autism spectrum disorders and schizophrenia. There are a number of relatively well-characterized factors that regulate synaptic strength. Many of these converge on the probability of neurotransmitter release and the amplitude of the postsynaptic response to the transmitter released by a single synaptic vesicle (quantal size). The purpose of this program is to explore a potentially fundamental but less well understood presynaptic factor-the amount of glutamate packaged per synaptic vesicle. This program focuses on the role of a novel Cl- conductance associated with the vesicular glutamate transporters (VGLUTs). The goal of this project is to determine the role of this conductance in the regulation of quantal size and excitatory transmission. The strategy is to determine the structural basis for this conductance and use this information to test its physiological role. Using Xenopus oocytes injected with mRNA constructs encoding internalization-defective VGLUTs, I will identify sequences responsible for this Cl- conductance and its allosteric activation. I will then rescue hippocampal neurons from VGLUT1/2 double knockout mice with wild type VGLUT2 and mutant forms with altered Cl- conductance. Using this strategy, I will determine the effects of the Cl- conductance on spontaneous and evoked release. The results will provide fundamental new insight into the regulation of quantal size, with important implications for excitatory neurotransmission.